AT 1 inhibition mediated neuroprotection after experimental traumatic brain injury is dependent on neutrophils in male mice

After traumatic brain injury (TBI) cerebral inflammation with invasion of neutrophils and lymphocytes is a crucial factor in the process of secondary brain damage. In TBI the intrinsic renin-angiotensin system is an important mediator of cerebral inflammation, as inhibition of the angiotensin II receptor type 1 (AT1) reduces secondary brain damage and the invasion of neutrophil granulocytes into injured cerebral tissue. The current study explored the involvement of immune cells in neuroprotection mediated by AT1 inhibition following experimental TBI. Four different cohorts of male mice were examined, investigating the effects of neutropenia (anti-Ly6G antibody mediated neutrophil depletion; C57BL/6), lymphopenia (RAG1 deficiency, RAG1−/−), and their combination with candesartan-mediated AT1 inhibition. The present results showed that reduction of neutrophils and lymphocytes, as well as AT1 inhibition in wild type and RAG1−/− mice, reduced brain damage and neuroinflammation after TBI. However, in neutropenic mice, candesartan did not have an effect. Interestingly, AT1 inhibition was found to be neuroprotective in RAG1−/− mice but not in neutropenic mice. The findings suggest that AT1 inhibition may exert neuroprotection by reducing the inflammation caused by neutrophils, ultimately leading to a decrease in their invasion into cerebral tissue.


Treatment. Application of antibodies for neutrophil granulocyte depletion and control antibodies.
For the depletion of neutrophils in WT mice (studies A and C) the Ly6G-specific antibody (anti-mouse, clone 1A8) was used. In the control antibody group, we used the isotype control antibody immunoglobulin IgG2a (rat, clone: 2A3). Both antibodies, anti-Ly6G (1A8) and IgG2a (2A3) (BXCell; West Lebanon, USA) were diluted in PBS with a final concentration of 2.5 mg/mL. We injected 0.2 mL (0.5 mg) of anti-Ly6G antibody (ND) and the same volume of the control IgG2a antibody (Ctrl) intraperitoneally (i.p.) 24 h before (studies A and C) and 24 h after experimental TBI (study C).
Study C: effect of AT1 inhibition in neutrophil depleted mice 3 days after TBI. Mice were randomized to treatment (24 h before and repeated 24 h after TBI) with either anti-Ly6G (ND) or IgG2a control antibody (Ctrl). They were subjected to CCI and then randomly assigned to additional treatment with candesartan (Cand) or vehicle solution (Veh), performed 30 min after TBI and then repeated daily, 24 and 48 h after TBI (Fig. 1). Therefore, the animals were randomly allocated to four treatment groups: Ctrl-Cand, Ctrl-Veh, ND-Cand and ND-Veh (n = 12/group). After the 72-h observation period, brains were removed for quantification of lesion volume, cytokine expression and activated microglia. Blood samples were withdrawn for hematological quantification of white blood cells (WBC), lymphocytes and neutrophils. For comparison we used naïve (non-operated) WT mice (n = 6; Fig. 1).  Figure 1. Experimental Timeline. Study C: Effect of AT1 inhibition in neutrophil depleted mice 3 days after TBI: C57BL/6 mice were randomized to treatment (24 h before and 24 h after TBI) with either anti-Ly6G or IgG2a control antibody. They were subjected to controlled cortical impact injury (CCI) and then randomly assigned to additional treatment with candesartan or vehicle solution, performed 30 min after TBI and then repeated daily, 24 and 48 h after TBI. Animals were randomly allocated to four treatment groups: (n = 12/group). To enhance the comparability of the effects on brain tissue infiltration dynamics between neutropenic and lymphopenic mice, we selected an observation time of 72 h after TBI. 72 h after CCI, brains were removed for quantification of lesion volume, cytokine expression and activated microglia (histology, PCR). Study D: Effect of AT1 inhibition in lymphopenic RAG1-deficient mice 3 days after TBI: RAG1-deficient mice (RAG1 −/− ) were randomly assigned to candesartan or vehicle solution treatment (n = 12/group) at 30 min, 24 and 48 h after CCI. 72 h after CCI, lesion volume, cytokine expression and activated microglia were quantified (histology, PCR). CCI, controlled cortical impact; N, neurological assessment; BW, body weight; BP, blood pressure; H, hematology (blood cell count); histology, lesion volume, activated microglia/macrophages; PCR, normalized (PPIA) gene expression (mRNA) of MPO, TNFα, TGFβ, IL1β, IL6 and iNOS. www.nature.com/scientificreports/ Study D: effect of AT1 inhibition in lymphopenic RAG1-deficient mice 3 days after TBI. RAG1-deficient mice were randomly assigned to candesartan or vehicle solution treatment (RAG1 −/− -Cand, RAG1 −/− -Veh; n = 12/ group) at 30 min, 24 and 48 h after TBI (Fig. 1). As in study C, 72 h after TBI, lesion volume, cytokine expression and activated microglia were quantified and hematologic assessment was performed. Additionally, we used naïve RAG1 −/− mice (n = 6; Fig. 1).
Measurement of physiological parameters. Before, and after experimental TBI body weight of each mouse was controlled. Blood pressure was measured 5 min before and after CCI under general anesthesia at the tail using a modified NIBP system (RTBP 2000, Kent Scientific, Torrington, USA; A/D converter: PCI 9112, Adlink Technology, Taiwan; software: Dasylab 5.0, measX, Germany; Flexpro 6.0, Weisang, Germany) as previously described 20 . Additionally, blood pressure values were determined in awake animals daily for 8 days before (training phase) and for 2 days after CCI. Perioperative body temperature was measured by a rectal temperature probe (Physitemp; Clifton, NJ, USA).
Assessment of functional outcome. In studies A, C and D neurological outcome was assessed using the rotarod performance test (Heidolph Instruments GmbH &Co.; Schwabach, Germany) as previously described [22][23][24][25] . After a pre-training phase (mice remained on a rotating rod for 20 s at 4 rpm) two days before TBI, the time to fall from the accelerating rod in the 2-min test period was registered. This test assesses coordination and motoric function and was performed 1 day before, 24 and 72 h after CCI. In study B functional outcome was determined by Neurological Severity Score 26 . In addition to the rotarod test, in studies C and D, functional outcome was also determined by modified neurological severity score (mNSS; modified after Tsenter et al. 26 ) 1 day before and 24 and 72 h after CCI 4 . To calculate mNSS, general behavior, alertness, motor ability and balance were rated with 6 different tasks. Each task was scored from 0 (normal) up to 3 (failed task). The mNSS ranges from 0 (healthy) to 16 (severely impaired) points 27 (Table 1). All neurological tests were performed by investigators blinded towards experimental group allocations.
Flow cytometry and blood cell count. At the end of observation period, great care was taken to perform accurate routine differential blood cell count. In deep anesthesia, EDTA anti-coagulated blood samples were taken from the retro-orbital veins as previously described 28 . The differential blood cell count was obtained via the ADVIA 2120i Hematology system by a medical technician specialized in murine blood analyses and blinded to experimental group allocation. The ADVIA 2120i is a Ly6G-independent full automated veterinary flow cytometry analyzer, validated for murine blood analyses. The analyses were performed after the standardized protocol of the Institute of Clinical Chemistry and Laboratory Medicine of the University Medical Center Table 1. Modified neurological severity score (mNSS). The modified Neurological Severity Score (mNSS) was designed on the basis of the Neurological Severity Score introduced by Tsenter et al. 26 . The mNSS focusses on motoric function and behavioral deficits and was performed 1 day before CCI and on posttraumatic day 1 and 3 (day 5 in study C) after experimental TBI. www.nature.com/scientificreports/ of Mainz (ADVIA 2120i Hematology System; Siemens Healthcare, Erlangen, Germany; https:// www. sieme nshealt hinee rs. com/ en-us/ hemat ology/ syste ms/ advia-2120-hemat ology-system-with-autos lide). The hematology analyzer ADVIA 2120i is a flow cytometry-based system that uses laser light scatter to differentiate and count WBC in two different ways: the peroxidase method and the lobularity/nuclear density method 29 . The peroxidase method uses the myeloperoxidase (MPO) to detect, differentiate and quantify the WBC when they pass through the flow cell. With the help of an optical system all WBC are counted, and peroxidase reagents are used to distinguish between MPO-positive cells, such as neutrophils, eosinophils, and monocytes, and peroxidase-negative cells, which include lymphocytes, and basophils 29 . The cells absorb light in proportion to the amount of peroxidase stain present, and this peroxidase activity parameter is represented on the x-axis of the peroxidase cytogram (Fig. 2a). Cells scatter light in proportion to their size, and this cell size parameter is represented on the y-axis of the cytogram (Fig. 2a). When the light-scatter and absorption data are plotted, distinct populations or clusters are formed, and cluster analysis is applied to identify different cell populations 29 . In the lobularity/nuclear density channel, surfactant and phthalic acid are used to lyse red blood cells and platelets, and to strip away the cytoplasmic membrane from all leukocytes, except basophils. Cells are then counted, and classified, according to size, lobularity, and nuclear density 29 . By the cluster analysis, the polymorphonuclear cells (neutrophils), the mononuclear leukocytes as well as the basophils are quantified.
Histologic and immunohistochemical evaluation. According to our previous protocol 4 brains were removed in deep anesthesia. For tissue evaluation, the brains were frozen in powdered dry ice and stored at − 20 °C. They were then cut in coronal plane with a cryostat (HM 560 Cryo-Star, Thermo Fisher Scientific, Walldorf, Germany) as previously described in detail 8 . The first slide was defined according to the first section corresponding to bregma + 3.14 mm in the Mouse Brain Library (www. mbl. org). 16 sections (12 and 20 µm) were collected at 500 µm-intervals, placed on Superfrost + TM slides (Thermo Fisher Scientific, Germany). In cresyl violet (Merck, Darmstadt, Germany) stained sections (12 µm), the total area of both hemispheres and the injured brain tissue area were determined for each section and animal using a computerized image analysis system (Delta Pix Insight; Maalov, Denmark) by an investigator blind to the group allocation. The total hemispheric brain volumes and the lesion volumes were calculated by following formula: 0.  4,8 . Immunohistochemical staining was performed as described before 27 . Briefly, cryosections (20 µm) were fixed in 4% paraformaldehyde in phosphate buffered saline (PBS), incubated with blocking solution (5% goat serum, 1% bovine serum albumin, and 0.1% TX-100 in PBS) for 1 h at room temperature. Primary antibodies specific for anti-ionized calcium-binding adapter molecule-1 (Iba-1; rabbit anti-mouse, anti-Iba-1 antibody; Wako Chemicals GmbH, Neuss, Germany), or Gr1 (rat anti-Ly6g + Ly6c, clone RB6-8C5, Abcam, UK) were applied in blocking solution overnight at 4 °C. The sections were washed, incubated with secondary biotin-conjugated antibodies (goat anti-rabbit IgG; Merck; Darmstadt, Germany) and processed according to the manufacturer's instructions using Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, USA), or fluorophore conjugated secondary antibodies (goat anti-rabbit IgG, Biotinylated; Merck; Darmstadt, Germany or goat anti-rat IgG Alexa Fluor 488, Thermo Fisher). Images of anti-Iba-1 immunostaining were taken at × 20 magnification (Axiovert, Zeiss, Germany), for anti-Gr-1 immunostaining at × 10 magnification (Keyence, BZ-X800). The total number of Iba-1-positive cells were counted at bregma − 1.28 mm in a region of interest (ROI) of 0.52 × 0.65 mm 2 in the cortical tissue adjacent to the lesion by an investigator blind to randomization, using ImageJ software (National Institutes of Health, USA). Iba-1-immunolabeled cells with appropriate morphology and appearance 30 were identified as activated microglia/macrophages and assessed in the ROI (0.52 × 0.65 mm 2 in the cortical tissue adjacent to the lesion). The rationale for counting Iba-1-positive cells in an area adjacent to the lesion rather than within the lesioned area was, that inside the lesion, where the tissue is essentially destroyed, microglia/macrophages are almost absent. In the perilesional area, however, there is a robust activation of microglia/macrophages. Results are presented in number of activated Iba-1 positive cells/mm 2 . Unfortunately, technical difficulties prevented us from obtaining an adequate quantitative assessment of Gr1-positive cells. Therefore, we present qualitative images with Gr1 staining (Fig. 2b).
Gene expression analysis. Brain tissue samples from the lesion and perilesional area of 500 µm coronal cryostat sections between histologic slice intervals were collected, snap frozen in liquid nitrogen, stored at − 80 °C. As described previously in detail 4,31 , after tissue sampling, extraction of mRNA and cDNA synthesis qPCR were performed (lysis: Qiazol-reagent, Qiagen, Hilden, Germany; homogenization: MM300 mill mixer, Retsch, Haan, Germany; RNA isolation: RNeasy Lipid Tissue Kit, Qiagen, Hilden, Germany; RNA concentration determined by spectrometer: NanoVue System, GE Healthcare Europe, Munich, Germany; RNA to cDNA reverse transcription Verso cDNA Kit, ABgene, Hamburg, Germany; cDNA amplification: real-time Ligthcycler 480 PCR System, Roche). PCR fragments of all applied genes were generated by PCR on an Eppendorf Thermocycler gradient (Eppendorf, Hamburg, Germany). The PCR products were purified with QIA quick PCR Purification Kit (Qiagen) according to the manufacturer´s instructions, and the DNA concentration was determined using NanoVue. A standard curve for absolute quantification was generated with PCR DNA for each PCR product (10 1 -10 7 DNA copies/µl), showing similar and good efficiency (90-110%; LightCycler Software, Roche) and linearity. Equal amounts of cDNA (1 µL) of each sample were analyzed in duplicates and amplified by realtime Lightcycler 480 PCR System (Roche). Real-time RT PCR kits were used according to the manufacturer´s instructions. All assays were conducted by an investigator blinded to group allocation. Using mouse-specific primers and probes ( Table 2) and optimized temperature conditions for qPCR, absolute copy numbers of the target genes, tumor necrosis factor α (TNFα), transforming growth factor β (TGFβ), interleukin 1β (IL1β), interleukin 6 (IL6), inducible nitric oxide synthase (iNOS) and myeloperoxidase (MPO) were calculated, and were then normalized against the absolute copy numbers of cyclophilin A (PPIA) 4 www.nature.com/scientificreports/ chosen as single normalizer 33 based on recent findings in our housekeeping gene study 32 . In order to improve comparability of the mRNA expression data between different treatment groups, and to eliminate qPCR kit dependent differences and limitations, qPCR data was normalized with PPIA and then related to normalized naïve target gene expression from naïve tissue samples from the corresponding brain region 34 . Therefore, normalized target gene expression values are expressed as % naïve expression 4 .

Statistical analysis.
All experiments were randomized and performed by investigators blinded toward the treatment groups (computer-based randomization: www. pubmed. de/ tools/ zufal lsgen erator). In order to determine the required sample size, the a priori power analysis using G * Power 35 was performed with the main variable, the primary endpoint, lesion volume data from previously published studies 4,8 . Therefore, based upon the data of these studies the present a priori power analysis was performed to determine an effect size of d 1.75, with an actual standard statistical power (1−β) of 0.95, and a significance level (α) of 0.05 and a sample size per group of n = 7. In order to have a sufficient power, we decided to have larger sample sizes (n = 8-12, per group) 36 . Statistical analysis was performed using the GraphPad Prism 8 Statistical Software (GraphPad Software Inc., La Jolla, CA, USA). Data distribution was tested by Shapiro-Wilks test. The comparisons of parametric and nonparametric data between two independent groups were done using the Welch-t test and the Wilcoxon rank sum test, respectively. For the statistical analysis of mNSS we performed ANOVA on ranks with the Kruskal-Wallis test, corrected for multiple comparisons using the Dunn's test. In this multi-arm parallel group randomized trial, for comparison of multiple independent groups, if the Shapiro-Wilk normality test was passed, one-way analysis of variance (one-way ANOVA) with post-hoc Holm-Šidák comparisons test (comparisons between all groups) was employed. In experimental groups where two separate treatment factors (neutrophil depletion and AT1 inhibition) are present, a two-way analysis of variance (two-way ANOVA) was performed. Physiologic data, blood cell count, lesion volumes, number of activated microglia and mRNA expression data were compared between experimental groups with two-way ANOVA and post hoc with all-pairwise multiple comparison procedures (Holm-Šidák method). To evaluate group differences in repeated measurements from the same animals (body weight, systolic blood pressure), repeated measures (RM) two-way ANOVA (two-factor repetition) was applied (factors: treatment and time), followed by Šidák's multiple comparisons test. Whenever there were missing values in the repeated measures dataset and a two-way ANOVA was not possible, repeated measures (mNSS, rotarod) data were analyzed with the mixed effect model using the restricted maximum likelihood (REML) method with Holm Šidák's multiple comparison test. The p values were adjusted for multiple comparisons. Values of p < 0.05 were considered significant. To identify outliers in our dataset, we employed a combination of the Figure 2. Depletion of neutrophils reduces brain damage after TBI (Study A). Male C57BL/6 mice were randomized to i.p. injection with specific anti-Ly6G (1A8) for neutrophil depletion (ND) or control antibody (Ctrl; IgG2a (2A3)) 24 h before TBI. With respect to the maximum brain tissue infiltration of neutrophils, lesion volume and cerebral inflammation were determined 4 h (ND-4h, Ctrl-4h; n = 6/group) and 24 h after TBI (ND-24h, Ctrl-24h; n = 8/group; p.i. = post injury). In diagrams control antibody IgG2a treated groups are depicted in white (Ctrl), neutrophil granulocyte depleted, anti-Ly6G treated groups are shown in light grey (ND). (a) Neutrophils in the WBC count: (a) top: representative cytograms of mice treated with control antibody IgG2a (Ctrl), and of mice treated with anti-Ly6G (ND) for neutrophil depletion. The cytograms, obtained with the ADVIA 2120i Hematology System contain the two channels of accurate quantification: peroxidase and basophil (lobularity/nuclear density

Results
Perioperative physiological parameters were stable in all groups. Peri-and intraoperative body temperature and systolic blood pressure were in all mice within physiological range, without considerable difference between groups ( Table 3). As published earlier, in our standardized anesthesia and operation setting, values were stable and within physiological limits 6 .
Low-dose candesartan treatment did not influence blood pressure after CCI. As the specific AT1 antagonist candesartan is used for the treatment of arterial hypertension, we determined its influence on arterial blood pressure. In the present study, low dose (0.1 mg/kg) candesartan treatment did not to alter blood pressure, as shown in previous studies 4,8 . During the observation period, in all groups, blood pressure was within physiological range (Table 3).
After TBI bodyweight was not affected by anti-Ly6G, RAG1-deficiency or AT1 inhibition. In naïve mice bodyweight was 24.7 ± 2.2 g. Before TBI, initial bodyweight was in Ctrl-4h mice 25.6 ± 0.9 and in ND-4h mice 25.5 ± 1.1 g. After 24 h, bodyweight was reduced in both Ctrl and ND, without any difference   Table 3. Systolic blood pressure after TBI. Systolic blood pressure [mmHg] during 36 posttraumatic hours was within physiologic range and not affected by low dose candesartan treatment. Time point 0 represents intraoperative measurement immediately after CCI induction under general anesthesia (in italic characters). At the following time points, measurements were performed in awake animals (normal characters). At certain time points there were significant differences, intergroup ( # p < 0.05 ND-Cand vs. Ctrl-Cand) and within the groups (ND-Veh: § p < 0.05 vs. 0 (CCI); ND-Cand: $ p < 0.05 vs. 6 h). www.nature.com/scientificreports/ (before: 25.8 ± 0.7 and 26.0 ± 1.2 g; 24 h after TBI: 24.1 ± 1.2* and 24.3 ± 1.3*, for Ctrl and ND, respectively, *p < 0.001 vs. before TBI). The body weight of RAG1 +/+ and RAG1 −/− were similarly reduced after CCI (24-h investigation: before: 22.7 ± 0.8 and 24.2 ± 1.9 g; 24 h after TBI: 20.3 ± 1.2* and 21.5 ± 1.7* g, for RAG1 +/+ and RAG1 −/− , respectively, *p < 0.001 vs. before TBI). In the 5-days investigation, after a body weight loss at 1 day after CCI, RAG1 +/+ and RAG1 −/− animals regained weight at 5 days after CCI in both groups (before TBI: 22.4 ± 1.1 and 22.2 ± 1.2 g, 1 day after TBI: 20.6 ± 1.2* # and 20.4 ± 0.5* # and 5 days after TBI: 22.3 ± 0.9 and 21.8 ± 1.5, for RAG1 +/+ and RAG1 −/− , respectively, *p < 0.001 vs. before and # p < 0.001 1 vs. 5 days after TBI). In both investigations RAG1 deficiency did not affect posttraumatic body weight loss. In all groups, there is a significant posttraumatic decrease of bodyweight, with a minimum on day 2 and an increase on day 3. However, neither neutrophil depletion, nor RAG1 deficiency, nor AT1 inhibition affected posttraumatic body weight loss, compared to control antibody, wild type or vehicle solution treated mice (Table 4).  www.nature.com/scientificreports/ Neutropenia was achieved with anti-Ly6G in WT, lymphopenia was present in RAG1 −/− mice. Differential blood cell counts were performed ( In ND-mice WBC reduction was more distinct and sustaining (ND-4h and-24h), with lasting relatively low WBC count to day 3 after TBI (ND-Veh and ND-Cand). In non-neutrophil-depleted mice, as a response to TBI, there was a shift from lymphocyte-dominated (84%) WBC to an elevation of the neutrophil fraction from 11% (naïve), to 43%, followed by a continuous decrease by 33% to 29 at 24 h (p < 0.05; Fig. 2a) and to 16% at 72 h in Ctrl-treated mice after TBI. In ND mice, in contrast, after TBI there is no initial elevation of neutrophils. Moreover, 24 and 72 h after TBI there is a significant neutropenia in ND-mice alongside elevated lymphocyte counts. Candesartan did not affect neutrophil counts (Table 5). Naïve RAG1 −/− mice are leukopenic, reduced lymphocyte counts (40%) are compensated by elevated neutrophil numbers (34%). After TBI in RAG1 −/− mice there is a decrease of WBC in vehicle treated mice, whereas in candesartan treated mice the decrease is not significant. In RAG1 −/− there is also a decrease of lymphocytes (25%) and an increase of neutrophils to 50%, after TBI, that are not affected by AT1 inhibition (Table 5). There is a sustaining posttraumatic decrease of monocytes in all groups. In candesartan treated ND mice, however there is a normalization of monocyte count 3 days after TBI. At 4 h after TBI there is a transient elevation of hemoglobin and hematocrit, with normalization of these parameters at 24 and 72 h after TBI. Platelets were within a physiological range in all groups at all time points and not affected by the treatment (Table 5).

Study A: effect of Neutrophil granulocyte depletion. Neutrophil depletion reduced tissue infiltration of neutrophils only at 4 h after TBI.
To detect the neutrophil infiltration into injured brain tissue we analyzed the gene expression of MPO, considered a neutrophil marker 37 , by qPCR (normalized naïve expression: 0.00000548 ± 0.00000017 mRNA/PPIA). In the first 4 h after TBI the gene expression of MPO is elevated to 159 ± 60% naïve in Ctrl mice. Neutrophil depletion by anti-Ly6G reduced MPO expression within the injured brain tissue by 56% (70 ± 24% naïve; p < 0.05), compared to Ctrl mice, at 4 h after CCI (Fig. 2b). However, at 24 h after TBI (i.e.: 48 h after application of anti-Ly6G), MPO gene expression of ND mice increased to the level of Ctrl (p < 0.05%; Fig. 2b), despite reduced neutrophils in the WBC count of ND mice (Fig. 2a).
Neutrophil granulocyte depletion did not affect neurological outcome. Neurological outcome was assessed before and 24 h after TBI by rotarod performance test. One day after TBI there was a marked neurological impairment in both groups (from 89 ± 15 and 78 ± 12 s before TBI to 38 ± 14 and 38 ± 8 s at 24 h after TBI, for Ctrl and ND, respectively, p < 0.001). However, neutrophil depletion did not alter neurological outcome compared to control group (Fig. 2f).

Study C: effect of Neutrophil granulocyte depletion combined with AT1 inhibition. AT1 in-
hibition had no effect on neutrophil blood cell count. To analyze an independent effect of candesartan treatment on neutrophil blood cell count we compared all treatment groups by two-way-ANOVA. While a sustained neutrophil depletion was achieved by anti-Ly6G (p < 0.001; Fig. 4a), AT1 inhibition did not affect neutrophil granulocyte count (Fig. 4a).
Neutrophil depletion and AT1 inhibition had no effect on neurological outcome. Neurological assessment was performed 1 day before, and 24 and 72 h after TBI using a mNSS and time spent staying on the rotarod. Compared to pre-trauma values, CCI induced a significant impairment in all experimental groups 24 h after TBI in mNSS (p < 0.001). Time spent on the rotarod and mNSS improved over time 3 days after TBI without differences between the treatment groups.
AT1 inhibition did not affect neurological outcome in RAG1-deficient mice. Neurological outcome was assessed by mNSS and rotarod at day 1 and 3 after TBI. There was a significant increase of neurological impairment (p < 0.001). However neurological deficit was not affected by candesartan treatment (Fig. 5d).

Discussion
To investigate the yet unexplained role of cellular immune response in the context of AT1 inhibition following TBI, we examined the impact of candesartan treatment in both neutrophil-depleted and lymphopenic RAG1 −/− mice. The current findings indicate that both neutropenia and lymphopenia independently contributed to a reduction in brain damage following TBI. AT1 inhibition after TBI resulted in a decrease in brain damage and neuroinflammation in Ctrl mice with normal neutrophil counts, as well as in lymphopenic RAG1 −/− mice. However, in neutrophil-depleted mice, AT1 inhibition had no effect on brain damage or neuroinflammation. Hence, the current findings indicate that the neuroprotective effects of AT1 inhibition are partially mediated by neutrophils.
To deplete neutrophils, we employed a specific antibody against Ly6G, which selectively targets neutrophils while leaving other cell types unaffected [40][41][42] . Numerous dosage concepts exist for administering anti-Ly6G (4-40 µg/g). We based our dosage decision (app. 20 µ/g) on murine studies that used anti-Ly6G to achieve sustained neutrophil depletion 41,43,44 . To ensure a significant reduction of neutrophils, that was monitored using the WBC count, we conducted preliminary pilot dosage finding studies. We found that the present dosage (500 µg anti-Ly6G), and the application interval (24 h before and after CCI) were effective in sustainingly reducing neutrophils in the WBC count for the entire 3-day observation period after TBI 41,43,45 . Furthermore, this dosage did not affect other blood cell populations, animal survival, or body weight. In contrast, the widely used less specific anti-Gr1 (clone RB6-8C5) not only reduces Ly6G-specific cells (neutrophils, Gr1+/Ly6G+), but also other lines of WBC with Ly6-receptors (dendritic cells and subpopulations of monocytes and CD8 T-lymphocytes) 40,46 .
To examine the effects of lymphopenia in TBI, we employed RAG1 deficient mice 47,48 . While RAG1 plays a key role in VDJ-recombination and B and T cell differentiation, RAG1 deficient mice have small lymphatic organs without mature B and T lymphocytes [47][48][49][50] . Their lymphocyte differentiation is blocked at an immature stage 49 . In the present study, RAG1 −/− showed to have lymphopenia. Although, certain cell lines, such as NK cells, may be present in RAG1 −/−51 , the lymphocytes detected in the WBC count of RAG1 −/− mice are likely immature lymphocytes 49 .
The selected observation periods for each study were based on the optimal timing for the maximum infiltration of the two immune cell types into the brain tissue. Neutrophils, the dominant blood cell population 24 h after TBI 52,53 , infiltrate early, with parenchymal infiltration peaking at 1 day after TBI 18,19 . From the third day on after TBI, lymphocytes invade into cerebral tissue 6,18 . To enhance the comparability of the impact of candesartan between the neutropenic and lymphopenic mice, we opted to utilize an observation period of 72 h following TBI in studies C and D, respectively 18 .
To ensure timely accurate hematologic analyses and interpretation and to minimize pre-analytic errors a mouse-species-appropriate practical hematologic instrumentation was performed with consistent collection method from the retroorbital sinus and a validated automated veterinary analyzer according to our standardized www.nature.com/scientificreports/ protocol 54,55 . The quality showed to be adequate and results are consistent with recent data 54 . Neutrophil depletion with anti-Ly6G may not completely eliminate circulating neutrophils that lack Ly6G expression on their surface, as previously noted by Boivin et al. 56 . However, our flow cytometry analysis was performed independently of Ly6G, as we used the ADVIA 2120i Hematology system for differential blood cell count 29 . This is a well-standardized flow cytometry-based system that distinguishes and counts WBC through two methods: peroxidase, detecting MPO-positive cells, and lobularity/nuclear density, classifying cells by size, lobularity, and nuclear density. Both methods allow for accurate identification and quantification of WBC populations, including neutrophils. Therefore, we believe that the presented neutropenia by anti-Ly6G treatment is accurate.
In the present study, we observed a transient leukopenia as part of the inflammatory reaction following TBI, which we attribute to trauma-associated neutrophil sequestration 57 . In Ctrl treated mice WBC count normalized one day after TBI. Anti-Ly6G treatment resulted in a sustained reduction of WBC count in mice after TBI, lasting up to 3 days (within physiological range) 54 . Neutrophils, the most common granulocytes, generally comprise 20-30% of WBC count in naïve wild type mice (70-80% are lymphocytes) 54 . In Ctrl mice, TBI caused a shift from lymphocyte-dominated WBC to a sustained and significant increase in neutrophil count 52,53 . ND mice experienced neutropenia and leukopenia in comparison to naïve mice, with an increase in lymphocyte count from 24 h post-TBI onwards. RAG1 −/− mice appeared to be leukopenic, and lymphopenia was compensated by elevated neutrophils. AT1 inhibition had no effect on posttraumatic blood cell count in wild type, neutropenic and RAG1 −/− mice.
In the present study the selective AT1 blocker candesartan was chosen, that crosses the BBB 16 . Consistent with prior research 4,8,12,17 , a low dose of candesartan (0.1 mg/kg) was administered to avoid any negative impact on blood pressure, which can exacerbate TBI outcomes 58,59 . To achieve sustained AT1 inhibition, treatment was started 30 min after TBI and then repeated daily 8 .
The peritraumatic body weight, a surrogate parameter of well-being and intake of food and water, was not affected by any treatment. The behavioral and neuromotor functions are impaired in this acute phase but show quick recovery in all groups. However, we did not detect any significant treatment effects on neurologic outcome using the NSS which was originally developed by Tsenter et al. 26 as a non-parametric assessment that focuses on motor function and behavior. Therefore, we tried to implement a more sensitive parameter for neurofunctional outcome, the Rotarod test, in subsequent studies. In studies C and D, we chose to combine the modified NSS (mNSS, adapted from Tsenter et al. 2008 4,26 ) with the Rotarod test in an attempt to detect any treatment effects on both motor function and behavior. Unfortunately, despite our efforts, we were not able to demonstrate any significant treatment effects on neurofunctional outcome. The current neurological assessments may lack sensitivity to detect the effects under investigation, or the protective effects on brain tissue may not have been significant enough to improve neurological deficits.
During the first days after TBI, microglia/macrophages show high phagocytic activity to remove necrotic and apoptotic cells, whereas astrocytes start to form a scar-like barrier around the brain lesion 60 . This leads to a contraction of the lesion-surrounding tissue and decrease of lesion volume assessed with Nissl staining. This can be observed in the present study considering different lesion sizes at different post-TBI time points, with cavitation processes beginning later than 5 days after CCI in our model (for example, at 7 days after CCI) 27 .
Consistent with recent research, our study demonstrated that neutrophil depletion decreased lesion size at 24 h, and neuroinflammation, following TBI 61 . However, neutrophil depletion did not affect lesion size at 72 h post-CCI in our study. In contrast, a recent study demonstrated sustained reduction of brain damage up to 14 days after CCI by using a less specific Gr1-antibody to achieve neutrophil depletion 61 . We hypothesize that neutrophils may affect brain injury at a very early time point after injury, as supported by previous live-imaging observations in mice 62 , and our results on MPO mRNA expression. We examined the gene expression of MPO, which is widely recognized as a reliable neutrophil marker 37,63,64 . MPO mRNA expression was reduced in the injured brain tissue of ND (compared to Ctrl) mice at 4 h after TBI, that is 28 h after application of anti-Ly6G. However, at later time points (24 h after CCI, 48 h after anti-Ly6G application), MPO mRNA expression increased in ND mice to the level of Ctrl mice. Thus, a reduction was not detectable concerning neutrophil brain tissue infiltration, despite reduced neutrophils in the WBC count of ND mice. In the present study anti-Ly6G was applied 24 h before CCI, and in Study C also at 24 h after CCI. Based on our findings that MPO expression was not different between ND and Ctrl mice at 48 h after the application of anti-Ly6G, we believe that brain tissue neutrophil infiltration is only reduced by anti-Ly6G in the first 24 h after the application, and therefore, at later time points there is no effect of antibody-mediated neutrophil depletion on parenchymal neutrophil infiltration. This is supported by recent reports that indicate that antibodies against neutrophil Ly6G do not inhibit neutrophil recruitment 42 . Our findings suggest that reduced neutrophil infiltration into damaged brain tissue within the first few hours after TBI (rather than after 24 h) results in beneficial effects, including reduced TNFα expression at 4 h, reduced structural brain damage at 24 h, and decreased microglial activation at 72 h post CCI.
While T cells are known to play diverse roles in adaptive immune responses and inflammation regulation, their specific role in TBI pathogenesis remains unclear 65 . Recent findings suggest that cerebral infiltration of T cells exacerbates neuroinflammation without affecting lesion volume after TBI 66,67 . A previous study on closedhead injury found no significant differences in pathological or neurological parameters between wild-type and RAG1 −/− mice up to 7 days post-injury 47 . The authors concluded that adaptive immunity is not crucial for initiating and sustaining inflammatory neuropathology after closed-head injury 47 . Another TBI study by Fee et al. showed that CD4 + T lymphocytes contribute to the severity of the acute phase of TBI and that brain injury is attenuated in RAG1 −/− mice compared to wild-type animals 48 . Consistent with these findings, RAG1 deficiency in the present study led to reduced brain damage at 1 and 5 days post-TBI. However, at 24 h post-TBI, compared to wild type, neutrophil depletion was found to be more effective in reducing lesion volume (33%) than lymphopenia (17%). Despite this, the reduction of lesion volume by lymphopenia was found to be more sustained (up to 5 days post-TBI) than that of neutropenia (only 24 h after TBI). The acute post-traumatic cerebral infiltration www.nature.com/scientificreports/ of neutrophils is more pronounced than that of lymphocytes 18 . Hence, it is conceivable that reducing neutrophil infiltration may have a more potent anti-inflammatory effect in the initial 24 h post-TBI than reducing lymphocyte infiltration, possibly explaining why brain damage and inflammation are consistently reduced in studies involving neutrophil depletion 61 , while results are inconsistent in studies involving RAG1 deficiency 47,48 . Emerging evidence indicates that the entire cellular immune response is modulated by the RAS 68,69 . AngII is a major mediator of cerebral inflammation and oxidative stress through AT1 7,70 . AT1 is widely expressed in the mature central nervous system, primarily in neurons, endothelial and smooth muscle cells, astrocytes, and microglia, which are important regulators of neuroinflammation 9,71 . AT1 is also expressed on migrating immune cells, like neutrophils, macrophages, and T-cells. AT1 activation triggers the production of chemokines, cytokines, and adhesion molecules, which promote the immigration of activated immune cells into the lesion site 7,[72][73][74][75] . This process induces inflammation and generates high levels of ROS via NADPH oxidase activation. AT1 signaling modulates NADPH oxidase complex activity and promotes the transcription of pro-inflammatory cytokines through activation of NF-κB dependent transcription 15,76 . Subsequently, AT1 activation stimulates various kinases, which propagate inflammatory responses and apoptotic pathways 17,[77][78][79][80] .
The present study demonstrated that repeated post-traumatic administration of candesartan in mice with normal post-traumatic neutrophil count led to reduced histological brain damage and decreased microglial activation at 3 days after TBI 8,12 . Candesartan treatment resulted in a 12% reduction in activated microglia/ macrophages, while neutrophil depletion led to a 8% reduction, compared to vehicle and control antibody treatment. These findings are consistent with earlier studies where candesartan treatment reduced the number of neutrophils and microglia/macrophages 3 days after TBI 4,17 .
Recent studies have demonstrated that AT1 inhibition after TBI reduces cytokine expression 7,8,81 . The pleiotropic cytokine TNFα is implicated in BBB dysfunction and the transmigration of WBC into brain tissue. It induces neuronal loss via microglial activation 2,82-84 . After an early upregulation in the first 8 h after TBI 83 TNFα decreases significantly in the following time 85 . This kinetic could explain that in the present study there is only a reduction of TNFα 4 h after TBI in ND mice. Cerebral IL1β expression increases in the first hour after TBI and reaches highest levels 12 and 24 h after experimental TBI 86 . Reduced activity of IL1β showed improved neurological outcomes and reduced infiltration of neutrophils 83,[87][88][89] . The absence of an effect on IL1β expression in the present study following neutropenia, RAG1 deficiency, or AT1 inhibition may be attributed to low levels of cytokine mRNA at 3 days post CCI 15 . Numerous studies have reported upregulation of IL6 following TBI, which is associated with increased microglial activation and neurological impairment 2,90 . Recent clinical studies suggest a correlation between elevated serum levels of IL6, increased ICP, and severity of TBI 91 . IL6 has also been shown to regulate the migration of neutrophils during acute inflammation 92 . In this study, AT1 inhibition led to a reduction in IL6 expression three days after TBI in Ctrl mice. However, candesartan did not affect IL6 expression in neutropenic mice.
Limitations of the present study are the short duration of reduced neutrophil infiltration into brain tissue, only detected at 4 h after CCI, and not at later time-points, as well as the absence of observed effects on neurological outcome. The fact, that we were not able to demonstrate the direct effect of AT1 inhibition on neutrophil infiltration into the brain tissue is a further limitation. Another significant limitation of the current study is the exclusive focus on acute post-traumatic time points. Further investigation is needed to examine the long-term impact of AT1 inhibition on structural brain damage, chronic brain inflammation, and cognitive dysfunction.
In a recent study, the protective effect of post-traumatic AT1 inhibition in young adult and aged mice was attributed to a decrease in microglia activation and an increase in anti-inflammatory microglia polarization. One major finding of the study was a significant reduction in neutrophil infiltration 4 . AT1 is expressed on both circulating neutrophils and lymphocytes 74,93 . However, in the previous study, perilesional T-cell immigration was not affected by AT1 inhibition 4 . The neuroprotective mechanisms of AT1 inhibition in the acute phase after TBI may thus not depend on the adaptive lymphocyte response. A recent study showed that expression of CD62L on human neutrophils is modulated by AT1 receptors, on various pathways involving ERK1/2, MAPK, and calcineurin 94 , leading to reduced transmigration of neutrophils. It has been shown that AT1 inhibition leads to down regulation of important recruitment proteins like ICAM1 in endothelial cells, and CD11b/CD18 on WBC, and reduces post traumatic increase of BBB permeability. Consequently, AT1 inhibition leads to a significant reduction in the infiltration of immune cells [95][96][97][98] . A recent murine cerebral transcriptomic analysis after TBI showed strong alterations of gene transcription, particularly of the innate immune response, by candesartan treatment 99 . Therefore, AT1 inhibition may have a direct and modulating anti-inflammatory effect on invading neutrophils and resident activated microglia 17 . AT1 inhibition appears to provide neuroprotection by reducing the inflammatory response of the innate immune system, as evidenced by reduced microglial activation and decreased infiltration of neutrophils. This suggests that the protective effect of AT1 inhibition is mediated by its anti-inflammatory properties 4 .

Conclusion
The present study indicates that the reduction of immune cells in both the innate and adaptive immune system, specifically neutropenia (ND), and lymphopenia (RAG1 −/− ), independently leads to decreased brain damage after TBI. Moreover, the study demonstrates that posttraumatic AT1 inhibition decreases brain damage and neuroinflammation in mice with normal neutrophil counts and in lymphopenic mice. However, in neutrophildepleted mice, AT1 blockage had no effect on brain damage and neuroinflammation. We conclude that the neuroprotective effects of AT1 inhibition are independent of lymphocytes but dependent on neutrophils. Thus, reduced neutrophil invasion into the injured brain tissue may mediate neuroprotection by AT1 inhibition. In summary, the study highlights the importance of immune cells in mediating neuroinflammation after TBI and the potential of AT1 inhibition as a therapeutic strategy against exacerbated neuroinflammation following TBI. www.nature.com/scientificreports/

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.